Method for the production of permanent human cell lines

Abstract

The present invention relates to a method for the production of a permanent human cell line, wherein isolated primary human cells are transfected simultaneously with a sequence allowing the expression of at least one cell transforming factor and a sequence allowing the expression of at least one recombinant polypeptide.

The invention claimed is: 
     
       1. A method for the production of a permanent human cell line comprising:
 (a) transfecting isolated primary human cells with a nucleic acid molecule or at least two different nucleic acid molecules, wherein in the case that one nucleic acid molecule is transfected, said nucleic acid molecule comprises a sequence or sequences encoding at least one cell transforming factor and an antibody, and wherein in the case that at least two different nucleic acid molecules are transfected, a first nucleic acid molecule comprises a sequence encoding at least one cell transforming factor, and a second nucleic acid molecule comprises a sequence encoding at least one antibody, wherein transfecting said first and second nucleic acid sequences occurs simultaneously; 
 (b) selecting said permanent human cell line expressing the antibody, wherein the selection is based on expression of said transforming factor and without any additional exogenous chemical selection. 
 
     
     
       2. The method according to  claim 1 , wherein the primary human cells are amniocytes, embryonic retina cells as well as embryonic cells of neuronal origin. 
     
     
       3. The method according to  claim 1 , wherein the cell transforming factors are T-antigen from SV40, E6 and E7 from HPV or E1A and E1B from human adenoviruses. 
     
     
       4. The method according to  claim 3 , wherein the sequence for E 1 A and E 1 B from human adenoviruses comprises the nucleotides 1 to 4344, 505 to 3522 or the nucleotides 505 to 4079 of the human adenovirus serotype 5. 
     
     
       5. The method according to  claim 1 , wherein, in case that the cell transforming factors are E6 and E7 from HPV, the sequences for E6 are present on one nucleic acid molecule and the sequences for E7 are present on another distinct nucleic acid molecule, and, in case that the cell transforming factors are E1A and E1B from human adenoviruses, the sequences for E1A are present on one nucleic acid molecule and the sequences for E1B are present on another distinct nucleic acid molecule. 
     
     
       6. The method according to  claim 5 , wherein sequences for the antibody are present on one of the two nucleic acid molecules for a cell transforming factor. 
     
     
       7. The method according to  claim 1 , wherein the sequence encoding the cell transforming factor or the antibody comprises a heterologous promoter and/or a heterologeous transcriptional termination element. 
     
     
       8. The method according to  claim 7 , wherein the promoter is the CMV (cytomegalovirus) promoter, EF-1α promoter, CAG promoter, human or murine pgk promoter, RSV promoter or SV40 promoter, and the transcriptional termination element is the polyadenylation sequences of the SV40 Large T-antigen or of the human G-CSF gene. 
     
     
       9. The method according to  claim 1 , wherein the sequence encoding the antibody comprises recognition sequences for recombinases, which recognition sequences flank the coding sequence of the antibody or the complete expression cassette of the antibody. 
     
     
       10. The method according to  claim 9 , wherein the recognition sequences are loxP, attB/attP or Frt sequences. 
     
     
       11. A permanent human cell line obtainable according to a method of  claim 1 .